Scand J Clin Lab Invest 1992; 52: 207-213
Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of British Columbia on 11/18/14 For personal use only.
Alteration of erythrocyte glutathione, cysteine and glutathione synthetase in alcoholic and non-alcoholic cirrhosis C. L O G U E R C I O , G. NARDI*
C.
DEL
VECCHIO
BLANCO,
M.
COLTORTI
&
Istituto di Medicina Generale e Metodologia Clinica, I Facolta di Medicina e Chirurgia, Univerita di Napoli and *Laboratorio di Biochimica, Stazione Zoologica’ Anton Dohrn’, Villa Comunale, Napoli, Italy.
Loguercio C, Del Vecchio Blanco C, Coltorti M, Nardi G. Alteration of erythrocyte glutathione, cysteine and glutathione synthetase in alcoholic and nonalcoholic cirrhosis. Scand J Clin Lab Invest 1992; 52: 207-213. Glutathione (GSH) and cysteine were determined in the plasma and the erythrocytes of alcoholic and non-alcoholic cirrhotics as fluorescent monobromobimane derivatives by high-performance liquid chromatography (HPLC). Cirrhotic patients displayed a significant decrease of plasma GSH, as well as of plasma cysteine, that was related to the degree of liver disease but not to the nutritional conditions. On the contrary, erythrocyte cysteine was found to increase significantly in all cirrhotics, particularly in alcoholics, regardless of the severity of disease. In an attempt to find a possible explanation of these alterations, the GSH synthesizing enzymes, y-glutamylcysteine synthetase (GC-s) and GSH synthetase (GSH-s) activities were determined in the erythrocytes. GSH-s activity was significantly lower in cirrhotic patients. whereas GC-s activity did not differ in the three groups.
Key words: alcohol; y-glutamylcysteine synthetase; liver; monobromobimanes Giovanna Nurdi, Luhorutorio rli Biochittiic.tr, Stuziotir Zoologicrr ‘Anton Dohrn’, Villa Comunule, 80121 Nupoli, Ituly
Glutathione (GSH) represents the major cellular low-molecular thiol involved in a variety of activities, including protection of cells from oxidative challenges, detoxification of xenobiotics and as a cysteine reservoir [l]. The total pool of plasma GSH is principally determined by the equilibrium between its synthesis and output by the liver and its degradation by the kidney [ 1,2]. Chronic liver injury is usually accompanied by hepatic and plasma GSH deficiency [3-51 and hepatic and plasma GSH decrease has also
been observed during chronic alcohol consumption [6-8].Recently, red blood cells in liver disease and alcoholism were also found to be modified in their low-molecular thiol contents. In this case GSH generally decreases whereas cysteine increases [SlO]. In the present study we have therefore evaluated plasma and erythrocyte GSH and cysteine of patients affected by liver cirrhosis with respect to (i) alcoholic or non-alcoholic aetiology and (ii) severity of liver disease and nutritional status. Furthermore, in an attempt 207
208
C . Loguercio et al.
to find a possible metabolic connection between the decrease of GSH and the increase of cysteine in the red blood cells in liver disease, the GSH synthesizing enzymes of erythrocytes were examined. PATIENTS AND METHODS
Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of British Columbia on 11/18/14 For personal use only.
Subjects The cirrhotic patients were divided into two groups: the first group consisting of 14 males and seven females affected by post-hepatitis cirrhosis, ages ranging between 39 and 62 years, median age 55 years; and the second group consisting of 25 males and two females with alcoholic cirrhosis, between 29 and 69 years of age, median age 56 years. The diagnosis of cirrhosis was based on laparoscopic and/or histological findings in 29 subjects. In the other patients whose blood coagulation parameters (prothrombin activity, number of platelets, etc.) did not allow this approach, the diagnosis was based on clinical, laboratory, ultrasonographic and upper gastrointestinal endoscopy. The degree of hepatic disease was evaluated according to Child-Pugh criteria [ll], consisting of clinical parameters (presence or absence of ascite and/or encephalopathy) and laboratory data (plasma bilirubin, prothrombin, albumin). The nutritional status was assessed by the evaluation of the skinfold thickness (biceps and triceps), according to Loguercio et al. [12]. Table I summarizes the features of the two groups. The control group consisted of 18 healthy volunteers (11 males and seven females), ages 23-63 years, median age 47 years, chosen
among the staff members of our Institute. None of the volunteers was an alcohol abuser. Ten subjects were non-drinkers and eight did not exceed 40 g ethanol daily. Liver damage was excluded on the basis of clinical, laboratory and ultrasonographic data. At least 2 days prior to the study, all pharmacological treatments (anti-aldosterone drugs, furosemide) were stopped, except lactulose for those who were receiving it. None of the patients received steroids and/or amino acids per 0s or per venam. All subjects were kept for at least 4 days prior to study on a normocaloric diet (35 Kcal kg-' body mass) containing 1 g kg-' of proteins, which was well tolerated by cirrhotics with encephalopathy. All subjects entering the study expressed informed consent. Chemicals Reduced GSH was from Sigma (St Louis, M O , USA) and monobromobimane (MB) was obtained with the name Thiolyte Reagent from Calbiochem (San Diego, C A , USA). All solvents for HPLC were from Fluka (Buchs, Switzerland). Determinations of GSH and cysteine in plasma and erythrocytes After an overnight fast, samples of venous blood (5 ml) were collected into 20 mmol I - ' EDTA and serine borate t o final a concentration of 20 mmol I - ' and put on ice. The samples to be derivatized with monobromobimane (MB) to give fluorescent compounds (250 pI of plasma or 50 pl of whole blood) were added
TABLE I . Principal features of cirrhotic patients (mean? SE)
Plasma A L T (Ref. v .
Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of British Columbia on 11/18/14 For personal use only.
Alteration of erythrocyte glutathione, cysteine and glutathione synthetase in alcoholic and non-alcoholic cirrhosis C. L O G U E R C I O , G. NARDI*
C.
DEL
VECCHIO
BLANCO,
M.
COLTORTI
&
Istituto di Medicina Generale e Metodologia Clinica, I Facolta di Medicina e Chirurgia, Univerita di Napoli and *Laboratorio di Biochimica, Stazione Zoologica’ Anton Dohrn’, Villa Comunale, Napoli, Italy.
Loguercio C, Del Vecchio Blanco C, Coltorti M, Nardi G. Alteration of erythrocyte glutathione, cysteine and glutathione synthetase in alcoholic and nonalcoholic cirrhosis. Scand J Clin Lab Invest 1992; 52: 207-213. Glutathione (GSH) and cysteine were determined in the plasma and the erythrocytes of alcoholic and non-alcoholic cirrhotics as fluorescent monobromobimane derivatives by high-performance liquid chromatography (HPLC). Cirrhotic patients displayed a significant decrease of plasma GSH, as well as of plasma cysteine, that was related to the degree of liver disease but not to the nutritional conditions. On the contrary, erythrocyte cysteine was found to increase significantly in all cirrhotics, particularly in alcoholics, regardless of the severity of disease. In an attempt to find a possible explanation of these alterations, the GSH synthesizing enzymes, y-glutamylcysteine synthetase (GC-s) and GSH synthetase (GSH-s) activities were determined in the erythrocytes. GSH-s activity was significantly lower in cirrhotic patients. whereas GC-s activity did not differ in the three groups.
Key words: alcohol; y-glutamylcysteine synthetase; liver; monobromobimanes Giovanna Nurdi, Luhorutorio rli Biochittiic.tr, Stuziotir Zoologicrr ‘Anton Dohrn’, Villa Comunule, 80121 Nupoli, Ituly
Glutathione (GSH) represents the major cellular low-molecular thiol involved in a variety of activities, including protection of cells from oxidative challenges, detoxification of xenobiotics and as a cysteine reservoir [l]. The total pool of plasma GSH is principally determined by the equilibrium between its synthesis and output by the liver and its degradation by the kidney [ 1,2]. Chronic liver injury is usually accompanied by hepatic and plasma GSH deficiency [3-51 and hepatic and plasma GSH decrease has also
been observed during chronic alcohol consumption [6-8].Recently, red blood cells in liver disease and alcoholism were also found to be modified in their low-molecular thiol contents. In this case GSH generally decreases whereas cysteine increases [SlO]. In the present study we have therefore evaluated plasma and erythrocyte GSH and cysteine of patients affected by liver cirrhosis with respect to (i) alcoholic or non-alcoholic aetiology and (ii) severity of liver disease and nutritional status. Furthermore, in an attempt 207
208
C . Loguercio et al.
to find a possible metabolic connection between the decrease of GSH and the increase of cysteine in the red blood cells in liver disease, the GSH synthesizing enzymes of erythrocytes were examined. PATIENTS AND METHODS
Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of British Columbia on 11/18/14 For personal use only.
Subjects The cirrhotic patients were divided into two groups: the first group consisting of 14 males and seven females affected by post-hepatitis cirrhosis, ages ranging between 39 and 62 years, median age 55 years; and the second group consisting of 25 males and two females with alcoholic cirrhosis, between 29 and 69 years of age, median age 56 years. The diagnosis of cirrhosis was based on laparoscopic and/or histological findings in 29 subjects. In the other patients whose blood coagulation parameters (prothrombin activity, number of platelets, etc.) did not allow this approach, the diagnosis was based on clinical, laboratory, ultrasonographic and upper gastrointestinal endoscopy. The degree of hepatic disease was evaluated according to Child-Pugh criteria [ll], consisting of clinical parameters (presence or absence of ascite and/or encephalopathy) and laboratory data (plasma bilirubin, prothrombin, albumin). The nutritional status was assessed by the evaluation of the skinfold thickness (biceps and triceps), according to Loguercio et al. [12]. Table I summarizes the features of the two groups. The control group consisted of 18 healthy volunteers (11 males and seven females), ages 23-63 years, median age 47 years, chosen
among the staff members of our Institute. None of the volunteers was an alcohol abuser. Ten subjects were non-drinkers and eight did not exceed 40 g ethanol daily. Liver damage was excluded on the basis of clinical, laboratory and ultrasonographic data. At least 2 days prior to the study, all pharmacological treatments (anti-aldosterone drugs, furosemide) were stopped, except lactulose for those who were receiving it. None of the patients received steroids and/or amino acids per 0s or per venam. All subjects were kept for at least 4 days prior to study on a normocaloric diet (35 Kcal kg-' body mass) containing 1 g kg-' of proteins, which was well tolerated by cirrhotics with encephalopathy. All subjects entering the study expressed informed consent. Chemicals Reduced GSH was from Sigma (St Louis, M O , USA) and monobromobimane (MB) was obtained with the name Thiolyte Reagent from Calbiochem (San Diego, C A , USA). All solvents for HPLC were from Fluka (Buchs, Switzerland). Determinations of GSH and cysteine in plasma and erythrocytes After an overnight fast, samples of venous blood (5 ml) were collected into 20 mmol I - ' EDTA and serine borate t o final a concentration of 20 mmol I - ' and put on ice. The samples to be derivatized with monobromobimane (MB) to give fluorescent compounds (250 pI of plasma or 50 pl of whole blood) were added
TABLE I . Principal features of cirrhotic patients (mean? SE)
Plasma A L T (Ref. v .
